One of the foremost barriers to cancer immunotherapy and immunoprevention is the phenomenon of tolerance, the immune system's safeguard against autoimmune disease. Most tumor antigens are self antigens showing little or no difference from their normal counterparts in amino acid sequence and three dimensional structure.
The immune system generally becomes tolerant to self antigens early in life. T lymphocyte clones specifically reactive to self antigens are either deleted or anergized during thymic development, or are kept in check at the periphery, mainly by diverse populations of regulatory T cells (Treg). Especially important are natural Treg which develop in the thymus upon high affinity recognition of antigens in the thymic stroma (Colombo and Piconese, 2007). It is often impossible to predict an antigen and immunization protocol that will break tolerance to a self antigen to achieve effective vaccination. This problem has defeated the development of many vaccines intended to induce immune response against tumor antigens (Wei et al, 2004).
A most promising tumor antigen in breast and other carcinomas is HER2 (ErbB-2, neu). HER2 is amplified in ˜30% of all breast cancers and is over-expressed in several other epithelial-derived neoplasms including ovarian cancer, small cell lung cancer, and cancers of the head and neck (Slamon, et al., 1989, Yu and Hung, 2000; Tzahar and Yarden. 1998).
HER2 receptors include an extracellular domain (ECD) of about 630 amino acids, a single membrane-spanning transmembrane region (TM), and an intracellular domain (ICD) including a cytoplasmic tyrosine kinase. The ECD contains four domains arranged as a tandem repeat of a two-domain unit consisting of a ˜190-amino acid L domain (domains I and III) followed by a ˜120-amino acid cysteine-rich domain (domains II and IV) (Witton, 2003, Roskoski, 2014).
Other members of the HER family of receptors, HER1, HER3, and HER4, bind extracellular growth factor (EGF) family ligands, but HER2 itself does not. Instead, it acts as a co-receptor, the preferred binding partner of the other HER family receptors. Ligand binding brings about heterodimerization of HER family receptors with HER2, leading to tyrosine kinase activation, and the activation of downstream signaling pathways. Overexpression of HER2, commonly seen in carcinomas, promotes spontaneous receptor dimerization and the activation of signaling pathways, in the absence of a ligand (Olayioye, 2001).
The presence of HER2 specific T cells and antibodies in breast and ovarian cancer patients indicate this molecule as a target of immunoprevention and therapy (Disis, et al., 1994; Peoples, et al., 1995; Fisk, et al., 1997; Kobayashi, et al., 2000). Passive immunotherapy, by administration of the anti-HER2 moAb (monoclonal antibody), Herceptin®, is used to treat patients with advanced breast cancer (Cobleigh, et al., 1999). Unfortunately, since ErbB-2 is a self antigen, and its sequence is typically unmodified in cancer, tumor hosts show strong immune tolerance against immune tolerance to HER2.
A HER2 tolerance breaking strategy that has shown some promise is to immunize a host with xenogeneic (heterologous) HER2, that is, HER2 from a different species than that of the immunized host. The strategy depends on the development of antigens that are sufficiently foreign to the HER2 of the host species to break tolerance to HER2, but sufficiently similar to elicit T cells and antibodies that cross react with the host HER2.
Some success has been attained with this strategy. It was found, for example, that heterologous vaccination with rat HER2 (rat neu) produced a degree of T cell response in human-HER2-tolerant transgenic mice. More complete responses were produced by vaccinating the transgenic mice with a hybrid antigen combining components of rat and human HER2 (Jacob, et al, 2006; Jacob, et al., 2010).
There is a need for more effective tolerance breaking antigens, for use in therapeutic and preventative vaccination against mammary carcinoma and other HER2-expressing cancers. There is also a need for monoclonal antibodies to such antigens, because such antibodies are themselves potential cross reacting reagents that can target HER2 expressing tumor cells.
There is also a need for antigens and methods useful for breaking tolerance to self HER2 in cats, for the treatment and prevention of mammary carcinoma in domestic feline populations. Feline mammary cancer is an important veterinary problem. The domestic cat population is estimated at 1 billion worldwide (Mullikin, et al., 2010) with approximately 95 million residing in US households (www.humanesociety.org/issues/pet_overpopulation/facts/pet_ownership_statistics.html) About 15% of unsprayed domestic cats spontaneously develop mammary tumors, 90% of which are malignant. Most of the malignancies are adenocarcinomas, with progression and histopathology similar to that of human breast cancer. HER2 expression has been reported in these tumors (Hayden, et al., 1971; Munson and Moresco, 2007; Gimenez, et al., 2010; Soares, et al., 2013; DeMaria, et al., 2005). Furthermore, successful HER2-targeted immunotherapies in outbred cat populations can lead directly to improved immunotherapies for human patients, which is not the case for immunotherapies developed with inbred rodent model populations. That is because the amino acid sequences of human and feline HER2 are more similar than those of human and mouse or rat neu (see, e.g., FIG. 6B), and because outbred cat populations exhibit a genetic diversity similar to that of human populations. There is therefore a need for antigens, vaccines, and methods for breaking tolerance to self HER2 for the therapeutic and preventative vaccination of mammary carcinomas of domestic cats.
Even with improved antigens and vaccines, a roadblock to breaking HER2 tolerance is the immunocompromised status of many cancer patients at the time of presentation for treatment. A competent immune system is required to meet the challenge of mounting a response to a self antigen. The induction of regulatory T cells, and the effects chemotherapy and radiation treatments can all contribute to a compromised immune system. There is a need for a diagnostic method for screening human and animal candidates for immunocompetence before the start of extended courses of tolerance-breaking immunotherapies.